Muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and methods of use thereof to target dystrophin and to treat Duchenne muscular dystrophy
Aspects of the disclosure relate to complexes comprising a muscle-targeting agent covalently linked to a molecular payload. In some embodiments, the muscle-targeting agent specifically binds to an internalizing cell surface receptor on muscle cells. In some embodiments, the molecular payload promotes the expression or activity of a functional dystrophin protein. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide, e.g., an oligonucleotide that causes exon skipping in a mRNA expressed from a mutant DMD allele.
1. A composition comprising complexes that comprise an anti-transferrin receptor antibody covalently linked to at least one exon-skipping oligonucleotide,
wherein the anti-transferrin receptor antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise human or humanized framework regions; wherein the anti-transferrin receptor antibody binds in the range of C89 to F760 of human transferrin receptor protein 1 (TfR1) having an amino acid sequence as set forth in SEQ ID NO: 1;
wherein each oligonucleotide of the complexes is 30 nucleotides in length and comprises a region of complementarity of 25, 26, 27, 28, 29, or 30 nucleotides in length, wherein the region of complementarity is complementary to the annealing site of an oligonucleotide consisting of the sequence of SEQ ID NO: 195, wherein each oligonucleotide of the complexes is a phosphorodiamidate morpholino oligomer (PMO); and
wherein, for each complex, each oligonucleotide of the complex is independently covalently linked at its 5′ end to a lysine residue of the anti-transferrin receptor antibody of the complex via a cleavable linker comprising a valine-citrulline sequence.
2. The composition of claim 1 , wherein the anti-transferrin receptor antibody is in the form of a Fab fragment.
3. The composition of claim 2 , wherein the region of complementarity is 30 nucleotides in length.
4. The composition of claim 3 , wherein each oligonucleotide of the complexes comprises 28, 29, or 30 contiguous nucleotides of the sequence of CTCCAACATCAAGGAAGATGGCATTTCTAG (SEQ ID NO: 296).
5. The composition of claim 4 , wherein, for each complex, the cleavable linker comprises a triazole covalently linked to the valine-citrulline sequence and further covalently linked to a lysine residue of the anti-transferrin receptor antibody,
wherein the triazole is obtained by a cycloaddition reaction between an azide and an alkyne to form the triazole, and
wherein prior to the cycloaddition reaction, the azide is covalently linked to the valine-citrulline sequence of the cleavable linker that is covalently linked to the 5′ end of the oligonucleotide of the complex, and the alkyne is provided in a bicyclononyne moiety.
6. The composition of claim 5 , wherein the anti-transferrin receptor antibody is not glycosylated.
7. The composition of claim 1 , wherein the nucleobases of the region of complementarity are selected from adenosine, thymidine, guanosine, and cytosine.
8. The composition of claim 1 , wherein the complexes of the composition on average comprise 1-3 exon-skipping oligonucleotides.
9. A method of inducing skipping of exon 51 of a DMD pre-mRNA in a muscle cell of a subject, the method comprising administering to the subject the composition of claim 1 .
10. A method of treating Duchenne muscular dystrophy in a subject in need thereof, the method comprising administering to the subject a composition comprising complexes that comprise an anti-transferrin receptor antibody covalently linked to at least one exon-skipping oligonucleotide,
wherein the anti-transferrin receptor antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise human or humanized framework regions; wherein the anti-transferrin receptor antibody binds in the range of C89 to F760 of human transferrin receptor protein 1 (TfR1) having an amino acid sequence as set forth in SEQ ID NO: 1;
wherein each oligonucleotide of the complexes is 30 nucleotides in length and comprises a region of complementarity of 25, 26, 27, 28, 29, or 30 nucleotides in length, wherein the region of complementarity is complementary to the annealing site of an oligonucleotide consisting of the sequence of SEQ ID NO: 195, wherein each oligonucleotide of the complexes is a phosphorodiamidate morpholino oligomer (PMO); and
wherein, for each complex, each oligonucleotide of the complex is independently covalently linked at its 5′ end to a lysine residue of the anti-transferrin receptor antibody of the complex via a cleavable linker comprising a valine-citrulline sequence.
11. The method of claim 10 , wherein each oligonucleotide of the complexes comprises the sequence of CTCCAACATCAAGGAAGATGGCATTTCTAG (SEQ ID NO: 296), and wherein the anti-transferrin receptor antibody is in the form of a Fab fragment.
12. The method of claim 11 , wherein, for each complex, the cleavable linker comprises a triazole covalently linked to the valine-citrulline sequence and further covalently linked to a lysine residue of the anti-transferrin receptor antibody,
wherein the triazole is obtained by a cycloaddition reaction between an azide and an alkyne to form the triazole, and
wherein prior to the cycloaddition reaction, the azide is covalently linked to the valine-citrulline sequence of the cleavable linker that is covalently linked to the 5′ end of the oligonucleotide of the complex, and the alkyne is provided in a bicyclononyne moiety.
13. The method of claim 10 , wherein the oligonucleotides of the complexes promote the expression or activity of a functional dystrophin protein in a muscle cell of the subject.
14. The method of claim 13 , wherein the muscle cell is a skeletal muscle cell or a cardiac muscle cell.
15. The method of claim 10 , wherein the nucleobases of the region of complementarity are selected from adenosine, thymidine, guanosine, and cytosine.
16. The method of claim 10 , wherein the subject is human.
17. The method of claim 10 , wherein the complexes of the composition on average comprise 1-3 exon-skipping oligonucleotides.
18. A composition comprising complexes that comprise an anti-transferrin receptor antibody covalently linked to at least one exon-skipping oligonucleotide,
wherein the anti-transferrin receptor antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise human or humanized framework regions; wherein the anti-transferrin receptor antibody binds in the range of C89 to F760 of human transferrin receptor protein 1 (TfR1) having an amino acid sequence as set forth in SEQ ID NO: 1;
wherein each oligonucleotide is 30 nucleotides in length, wherein each oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO) comprising the sequence of CTCCAACATCAAGGAAGATGGCATTTCTAG (SEQ ID NO: 296); and
wherein, for each complex, each oligonucleotide is independently covalently linked at its 5′ end to a lysine residue of the anti-transferrin receptor antibody via a cleavable linker comprising a valine-citrulline sequence.
19. The composition of claim 18 , wherein the anti-transferrin receptor antibody is in the form of a Fab fragment.
20. The composition of claim 19 , wherein the cleavable linker comprises a triazole covalently linked to the valine-citrulline sequence and further covalently linked to a lysine residue of the anti-transferrin receptor antibody,
wherein the triazole is obtained by a cycloaddition reaction between an azide and an alkyne to form the triazole, and
wherein prior to the cycloaddition reaction, the azide is covalently linked to the valine-citrulline sequence of the cleavable linker that is covalently linked to the 5′ end of the oligonucleotide and the alkyne is provided in a bicyclononyne moiety.
21. The composition of claim 20 , wherein the anti-transferrin receptor antibody is not glycosylated.
22. The composition of claim 18 , wherein the complexes of the composition on average comprise 1-3 exon-skipping oligonucleotides.
23. A method of inducing skipping of exon 51 of a DMD pre-mRNA in a muscle cell of a subject, the method comprising administering to the subject the composition of claim 18 .
24. A method of treating Duchenne muscular dystrophy in a subject in need thereof, the method comprising administering to the subject a composition comprising complexes that comprise an anti-transferrin receptor antibody covalently linked to at least one exon-skipping oligonucleotide,
wherein the anti-transferrin receptor antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise human or humanized framework regions; wherein the anti-transferrin receptor antibody binds in the range of C89 to F760 of human transferrin receptor protein 1 (TfR1) having an amino acid sequence as set forth in SEQ ID NO: 1;
wherein each oligonucleotide is 30 nucleotides in length, wherein each oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO) comprising the sequence of CTCCAACATCAAGGAAGATGGCATTTCTAG (SEQ ID NO: 296); and
wherein, for each complex, each oligonucleotide is independently covalently linked at its 5′ end to a lysine residue of the anti-transferrin receptor antibody via a cleavable linker comprising a valine-citrulline sequence.
25. The method of claim 24 , wherein the cleavable linker comprises a triazole covalently linked to the valine-citrulline sequence and further covalently linked to a lysine residue of the anti-transferrin receptor antibody,
wherein the triazole is obtained by a cycloaddition reaction between an azide and an alkyne to form the triazole, and
wherein prior to the cycloaddition reaction, the azide is covalently linked to the valine-citrulline sequence of the cleavable linker that is covalently linked to the 5′ end of the oligonucleotide and the alkyne is provided in a bicyclononyne moiety.
26. The method of claim 25 , wherein the anti-transferrin receptor antibody is not glycosylated.
27. The method of claim 24 , wherein the oligonucleotides promote the expression or activity of a functional dystrophin protein in a muscle cell of the subject.
28. The method of claim 27 , wherein the muscle cell is a skeletal muscle cell or a cardiac muscle cell.
29. The method of claim 24 , wherein the subject is human.
30. The method of claim 24 , wherein the complexes of the composition on average comprise 1-3 exon-skipping oligonucleotides.